The objective of this Phase I 2nd resubmitted proposal is to demonstrate the feasibility of establishing a screening assay and platform (SP-qNPA Microfluidic Screening Platform) that monitors host cell response to detect exposure to, and provide diagnosis of wellness and potentially specific infectious agents (NIAID category A, B and C pathogens), determine the stage of infection, and either indicate patients to be isolated and followed up with more testing, or even permit selection of the best treatment and monitoring of treatment course. The screening platform will perform a novel array-based multiplexed assay of gene expression (the quantitative Nuclease Protection Assay, qNPA"). The assay will be performed on a microfluidic platform. For Phase I that microfluidic platform is an adaptation of a current operational system that currently performs PCR and ELISA assays, developed by our collaborator, the Applied Nanobioscience Center (ANBC) of the BioDesign Institute, Arizona State University. A programmable open format universal array that can be changed to measure any set of molecules by the simple addition of an appropriate reagent will be used to perform the screening SP-qNPA assay. The platform will provide a fast turn around time (TAT) and enable the use of standard clinical samples (anti-coagulated blood, fixed tissue), not just samples specially prepared for the measurement of RNA. The assays of gene expression and protein only require lysis of the sample. Thus, this approach eliminates the need to extract RNA or carry out any biosynthetic step, such as reverse transcription or gene amplification, in order to measure RNA, greatly simplifying the assay. The sensitivity will be <3,000 target RNA molecules and <1ng/ml protein. The assays will be developed and integrated into the microfluidic platform as a b-test breadboard to permit performance to be determined as go/no-go criteria to proceed with a Phase II proposal. Thus, Phase I is designed to validate performance and address the major risk factors involved in the implementation of the qNPA to measure gene expression. Adaptation of the microfluidic compatable qNPA will be developed first in the same microplate format as the current commercial ArrayPlate qNPA is performed, benchmarked against the current assay, and then transferred into the microfluidic hardware for optimization, determination of go/no-go performance criteria, and further benchmarking against the current ArrayPlate qNPA. Ultimately, it is anticipated that the screening platform hardware costs will be <$2,000, the cost/test will be <$5 each, and the platform will be compact, easily transported, battery powered, robust, and capable of carrying out new response just-in-time tests simply by formatting different reagents. This screening platform will permit novel diagnostics to be developed that are useful in screening blood donations, and possibly for identifying and staging infectious diseases and all types of progressive disorders and to provide early diagnosis of the onset of adverse events such as transplant rejection or monitoring the onset of flares in lupus, as well as for identifying exposure to chemical agents and radiation using standard clinical samples. [unreadable] [unreadable] [unreadable]